In our Phase 1 project we have developed R-type pyocins, targetable bactericidal proteins, that kill the plague bacterium, Yersinia pestis, in vitro and are not sensitive to the mechanisms that bacteria use to resist antibiotics. Thus, these agents are candidates for protecting or treating humans exposed to plague, particularly strains that have been engineered to be broadly resistant to traditional antibiotics. The same R-type pyocins that can kill Y. pestis in vitro have efficacy when administered intraperitoneally or intravenously to mice infected systemically with Pseudomonas aeruginosa. This Phase 2 application proposes to characterize the immunogenicity of R-type pyocins in mice and then determine whether PEGylation of pyocins can reduce their immunogenicity without compromising potency. In addition, we shall complete pharmacokinetic (PK) characterizations of R-type pyocins in mice and rats and establish antimicrobial pharmacodynamic (PD) parameters in mice. These latter studies are necessary to generate PK and PD parameters to guide an efficacy study of R-type pyocins in mice with virulent pneumonic plague under BSL-3 containment. Allometric scaling parameters derived from the PK studies in 2 rodent species will provide guidance for the eventual Phase 1 IND safety and PK studies in humans, not part of this application. The proposed studies will provide critical information about: (1) the immunogenicity of R-type pyocins in mice and modulation of their immunogenicity by selective PEGylation. (2) the pharmacokinetic parameters of these potent bactericidal proteins in mice and rats (3) the antimicrobial pharmacodynamic parameters of R-type pyocins in mice lethally infected with Pseudomonas aeruginosa (4) the efficacy of R-type pyocins in murine pneumonic plague. PUBLIC HEALTH RELEVANCE: Yersinia pestis (plague bacteria) is a Category A pathogen that is deemed a significant threat to the U.S. as a biowarfare agent. If weaponized in an aerosol form, plague can easily gain access to the human respiratory system to cause pneumonia, shock and death within one day. Pneumonic plague, with its rapid progression and fatality rates over 80%, is much more difficult to treat than bubonic plague. Furthermore, this form of plague is highly contagious, being easily passed from human to human or animal to human. Because much of the biology of how bacteria develop drug resistance now is known and a clinical isolate of Y. pestis resistant to 9 antibiotics has been described, weaponized plague bacteria resistant to most front-line antibiotics is a real threat, i.e. a superweapon. We have discovered and designed R-type pyocins that kill plague bacteria in the tube and are not sensitive to the multiple mechanisms bacteria deploy to resist antibiotics. We propose to conduct the pharmacologic studies necessary for the development of R-type pyocins as targetable bactericidal proteins against plague and to demonstrate the efficacy of these R-type pyocins in an animal model of pneumonic plague.